DNA Damage Repair Inhibitors for Treatment of Cancer

ABSTRACT

The present invention relates to the recognition that inhibition of the base excision repair pathway is selectively lethal in cells which are deficient in HR dependent DNA DSB repair. Methods and means relating to the treatment of cancers which are deficient in HR dependent DNA DSB repair using inhibitors which target base excision repair components, such as PARP, is provided herein.

CROSS REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 13/286,475 filed Nov. 1, 2011, which is a Continuation ofapplication Ser. No. 11/001,474 filed Nov. 30, 2004, now U.S. Pat. No.8,071,574 issued Dec. 6, 2011, which claims benefit of U.S. ProvisionalPatent Application No. 60/526,244, filed Dec. 1, 2013. This applicationclaims benefit to GB0327844.7 filed Dec. 1, 2013. The applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

This invention relates to the induction of cellular lethality in cancercells, in particular cancer cells that are deficient in homologousrecombination (HR) dependent DNA double strand break (DSB) repair.

The effective repair of DNA damage in cells relies on mechanisms ofdamage sensing followed by the transduction of damage signals todownstream effectors that arrest at cell cycle checkpoints and repairDNA damage. Cells contain a number of distinct pathways of signals andeffectors that mediate the repair of different types of DNA damage.These pathways include base excision repair (BER), homologousrecombination (HR) dependent DNA double strand break (DSB) repair,non-homologous end joining (NHEJ), nucleotide excision repair (NER),base excision repair (BER) and mismatch repair (MMR). The interactionand interdependence between the various DNA repair pathways remainspoorly understood.

SUMMARY OF THE INVENTION

The present inventors have discovered that the inhibition of the BERpathway, for example by inhibition of poly (ADP-ribose) polymerase(PARP), is selectively lethal to those cancer cells that are deficientin HR dependent DNA DSB repair pathway. This has important implicationsin the treatment of cancer conditions.

One aspect of the invention provides the use of an inhibitor of a baseexcision repair pathway in the manufacture of a medicament for use inthe treatment of cancer in an individual, wherein said cancer isdeficient in HR dependent DNA DSB repair activity.

A method of treatment of cancer in an individual may comprise:administering an inhibitor of a base excision repair pathway to saidindividual, wherein said cancer is deficient in the HR dependent DNA DSBrepair pathway.

The cancer may comprise one or more cancer cells having a reduced orabrogated ability to repair DNA by said second repair pathway relativeto normal cells.

The HR dependent DNA DSB repair pathway repairs double-strand breaks(DSBs) in DNA via homologous mechanisms to reform a continuous DNA helix(K. K. Khanna and S. P. Jackson, Nat. Genet. 27(3): 247-254 (2001)). Thecomponents of the HR dependent DNA DSB repair pathway include ATM(NM_000051), RAD51 (NM_002875), RAD51L1 (NM_002877), RAD51C (NM_002876),RAD51L3 (NM_002878), DMC1 (NM_007068), XRCC2 (NM_005431), XRCC3(NM_005432), RAD52 (NM_002879), RAD54L (NM_003579), RAD54B (NM_012415),BRCA1 (NM_007295), BRCA2 (NM_000059), RAD50 (NM_005732), MRE11A(NM_005590) and NBS1 (NM_002485); where the designations refer toGenbank accession numbers. Other proteins involved in the HR dependentDNA DSB repair pathway include regulatory factors such as EMSY(Hughes-Davies et al, Cell, Vol 115, pp 523-535).

The base excision repair (BER) pathway repairs DNA single strand breaksand gaps and removes specific damaged bases. Gaps in the DNA helix arefilled by the sequential action of a Poly(ADP-Ribose) Polymerase (PARP)and a ligase. (K. K. Khanna and S. P. Jackson, Nat. Genet., 27(3):247-254 (2001); F. Dantzer et al. Biochemistry 39, 7559-69 2000; J. H.Hoeijmakers, Nature 411 366-74 (2001)). An inhibitor of base excisionrepair may inhibit any one of the components of the base excision repairpathway. Components of the BER pathway include: UNG (NM_003362), SMUG1(NM_014311), MBD4 (NM_003925), TDG (NM_003211), OGG1 (NM_002542), MYH(NM_012222), NTHL1 (NM_002528), MPG (NM_002434), NEIL1 (NM_024608),NEIL2 (NM_145043), NEIL3 (NM_018248), APE1 (NM_001641), APE2(NM_014481), LIG3 (NM_013975), XRCC1 (NM_006297), ADPRT (PARP1)(NM_0016718) and ADPRTL2 (PARP2) (NP_005475).

BER inhibitors may be used in the treatment of HR dependent DNA DSBrepair deficient cancers in combination with a DNA damaging agent.Preferably, the DNA damaging agent is used in a dosage or formulationthat, in the absence of the BER inhibitor, is not lethal to cells.Suitable DNA damaging chemotherapeutic agents are described below.

In some preferred embodiments, an inhibitor of the mammalian enzyme poly(ADP-ribose) polymerase (PARP) (D'Amours et al, (1999) Biochem. J. 342:249-268) may be employed. A PARP inhibitor may thus be used for thetreatment of a cancer which is deficient in HR dependent DNA DSB repair.

A method of treatment of a cancer deficient in HR dependent DNA DSBrepair in an individual may comprise:

administering a PARP inhibitor to said individual.

A PARP inhibitor may be used in the manufacture of a medicament for usein the treatment of cancer in an individual, wherein said cancer isdeficient in HR dependent DNA DSB repair.

PARP inhibitors are described in more detail below.

A cancer which is deficient in HR dependent DNA DSB repair may compriseor consist of one or more cancer cells which have a reduced or abrogatedability to repair DNA DSBs through that pathway, relative to normalcells i.e. the activity of the HR dependent DNA DSB repair pathway maybe reduced or abolished in the one or more cancer cells.

The activity of one or more components of the HR dependent DNA DSBrepair pathway may be abolished in the one or more cancer cells of anindividual having a cancer which is deficient in HR dependent DNA DSBrepair. Components of the HR dependent DNA DSB repair pathway are wellcharacterised in the art (see for example, Wood et al (2001) Science 2911284-1289) and include the components listed above.

In some preferred embodiments, the cancer cells may have a BRCA1 and/ora BRCA2 deficient phenotype i.e. BRCA1 and/or BRCA2 activity is reducedor abolished in the cancer cells. Cancer cells with this phenotype maybe deficient in BRCA1 and/or BRCA2 i.e. expression and/or activity ofBRCA1 and/or BRCA2 may be reduced or abolished in the cancer cells, forexample by means of mutation or polymorphism in the encoding nucleicacid, or by means of mutation or polymorphism in a gene encoding aregulatory factor, for example the EMSY gene which encodes a BRCA2regulatory factor (Hughes-Davies et al, Cell, Vol 115, pp 523-535).

BRCA1 and BRCA2 are known tumour suppressors whose wild-type alleles arefrequently lost in tumours of heterozygous carriers (Jasin M. Oncogene.2002 Dec. 16; 21(58):8981-93; Tutt et al Trends Mol Med.(2002)8(12):571-6). The association of BRCA1 and/or BRCA2 mutations withbreast cancer is well-characterised in the art (Radice P J Exp ClinCancer Res. 2002 September; 21(3 Suppl):9-12). Amplification of the EMSYgene, which encodes a BRCA2 binding factor, is also known to beassociated with breast and ovarian cancer.

Carriers of mutations in BRCA1 and/or BRCA2 are also at elevated risk ofcancer of the ovary, prostate and pancreas.

In some embodiments, a cancer condition in an individual may have beenpreviously identified as a cancer which is deficient in HR dependent DNADSB repair.

In other embodiments, a method as described herein may comprise the stepof identifying a cancer condition in an individual as deficient in HRdependent DNA DSB repair.

A cancer may be identified as a HR dependent DNA DSB repair deficientcancer, for example, by determining the activity of the HR dependent DNADSB repair pathway in one or more cancer cells from a sample obtainedfrom the individual or by determining the activity of one or morecomponents of the pathway. Activity may be determined relative to normal(i.e. non-cancer) cells, preferably from the same tissue.

The activity of the HR dependent DNA DSB repair pathway may bedetermined by measuring the formation of foci containing Rad51 in thenucleus in response to DNA damaging agents or PARP inhibitors. Cellsdeficient in the HR dependent DNA DSB repair pathway lack the ability toproduce such foci. The presence of Rad51 foci may be determined usingstandard immunofluorescent techniques.

Other methods for determining the activity of the HR dependent DNA DSBrepair pathway may include sensitivity to IR, chemotherapeutics such asinter-strand cross linking reagents, DSB inducing agents (TopoisomeraseI & II inhibitors) as well as the use of western blot analysis,immunohistology, chromosomal abnormalities, enzymatic or DNA bindingassays and plasmid-based assays.

In some embodiments, a cancer may be identified as deficient in an HRdependent DNA DSB repair pathway by determining the presence in cancercells from the individual of one or more variations, for example,polymorphisms or mutations, in a nucleic acid encoding a polypeptidewhich is a component of the HR dependent DNA DSB repair pathway.

Sequence variations such as mutations and polymorphisms may include adeletion, insertion or substitution of one or more nucleotides, relativeto the wild-type nucleotide sequence. In some embodiments, the variationmay be a gene amplication, for example an amplification of the EMSY gene(CAD22881; gene symbol C11ORF30). The one or more variations may be in acoding or non-coding region of the nucleic acid sequence and, may reduceor abolish the expression or function of the HR dependent DNA DSB repairpathway component polypeptide. In other words, the variant nucleic acidmay encode a variant polypeptide which has reduced or abolished activityor may encode a wild-type polypeptide which has little or no expressionwithin the cell, for example through the altered activity of aregulatory element. A variant nucleic acid may have one, two, three,four or more mutations or polymorphisms relative to the wild-typesequence.

The presence of one or more variations in a nucleic acid which encodes acomponent of the HR dependent DNA DSB repair pathway, may be determinedby detecting, in one or more cells of a test sample, the presence of anencoding nucleic acid sequence which comprises the one or more mutationsor polymorphisms, or by detecting the presence of the variant componentpolypeptide which is encoded by the nucleic acid sequence.

Various methods are available for determining the presence or absence ina sample obtained from an individual of a particular nucleic acidsequence, for example a nucleic acid sequence which has a mutation orpolymorphism that reduces or abrogates the expression or activity of aHR dependent DNA DSB repair pathway component. Furthermore, havingsequenced nucleic acid of an individual or sample, the sequenceinformation can be retained and subsequently searched without recourseto the original nucleic acid itself. Thus, for example, scanning adatabase of sequence information using sequence analysis software mayidentify a sequence alteration or mutation.

Methods according to some aspects of the present invention may comprisedetermining the binding of an oligonucleotide probe to nucleic acidobtained from the sample, for example, genomic DNA, RNA or cDNA. Theprobe may comprise a nucleotide sequence which binds specifically to anucleic acid sequence which contains one or more mutations orpolymorphisms and does not bind specifically to the nucleic acidsequence which does not contain the one or more mutations orpolymorphisms, or vice versa.

The oligonucleotide probe may comprise a label and binding of the probemay be determined by detecting the presence of the label.

A method may include hybridisation of one or more (e.g. two)oligonucleotide probes or primers to target nucleic acid. Where thenucleic acid is double-stranded DNA, hybridisation will generally bepreceded by denaturation to produce single-stranded DNA. Thehybridisation may be as part of a PCR procedure, or as part of a probingprocedure not involving PCR. An example procedure would be a combinationof PCR and low stringency hybridisation.

Binding of a probe to target nucleic acid (e.g. DNA) may be measuredusing any of a variety of techniques at the disposal of those skilled inthe art. For instance, probes may be radioactively, fluorescently orenzymatically labelled. Other methods not employing labelling of probeinclude examination of restriction fragment length polymorphisms,amplification using PCR, RN′ase cleavage and allele specificoligonucleotide probing. Probing may employ the standard Southernblotting technique. For instance, DNA may be extracted from cells anddigested with different restriction enzymes. Restriction fragments maythen be separated by electrophoresis on an agarose gel, beforedenaturation and transfer to a nitrocellulose filter. Labelled probe maybe hybridised to the DNA fragments on the filter and binding determined.

Those skilled in the art are well able to employ suitable conditions ofthe desired stringency for selective hybridisation, taking into accountfactors such as oligonucleotide length and base composition, temperatureand so on.

Suitable selective hybridisation conditions for oligonucleotides of 17to 30 bases include hybridization overnight at 42° C. in 6×SSC andwashing in 6×SSC at a series of increasing temperatures from 42° C. to65° C.

Other suitable conditions and protocols are described in MolecularCloning: a Laboratory Manual: 3rd edition, Sambrook & Russell (2001)Cold Spring Harbor Laboratory Press NY and Current Protocols inMolecular Biology, Ausubel et al. eds. John Wiley & Sons (1992).

Nucleic acid, which may be genomic DNA, RNA or cDNA, or an amplifiedregion thereof, may be sequenced to identify or determine the presenceof polymorphism or mutation therein. A polymorphism or mutation may beidentified by comparing the sequence obtained with the database sequenceof the component, as set out above. In particular, the presence of oneor more polymorphisms or mutations that cause abrogation or loss offunction of the polypeptide component, and thus the HR dependent DNA DSBrepair pathway as a whole, may be determined.

Sequencing may be performed using any one of a range of standardtechniques. Sequencing of an amplified product may, for example, involveprecipitation with isopropanol, resuspension and sequencing using aTaqFS+ Dye terminator sequencing kit. Extension products may beelectrophoresed on an ABI 377 DNA sequencer and data analysed usingSequence Navigator software.

A specific amplification reaction such as PCR using one or more pairs ofprimers may conveniently be employed to amplify the region of interestwithin the nucleic acid sequence, for example, the portion of thesequence suspected of containing mutations or polymorphisms.

The amplified nucleic acid may then be sequenced as above, and/or testedin any other way to determine the presence or absence of a mutation orpolymorphism which reduces or abrogates the expression or activity ofthe HR dependent DNA DSB repair pathway component.

Suitable amplification reactions include the polymerase chain reaction(PCR) (reviewed for instance in “PCR protocols; A Guide to Methods andApplications”, Eds. Innis et al, 1990, Academic Press, New York, Mulliset al, Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich(ed), PCR technology, Stockton Press, N Y, 1989, and Ehrlich et al,Science, 252:1643-1650, (1991)).

In some embodiments, a cancer may be identified as deficient in a HRdependent DNA DSB repair by assessing the level of expression oractivity of a positive or negative regulator of a component of the HRdependent DNA DSB repair pathway, such as EMSY. Expression levels may bedetermined, for example, by Western blot, ELISA, RT-PCR, nucleic acidhybridisation or karyotypic analysis.

In some preferred embodiments, the individual is heterozygous for one ormore variations, such as mutations and polymorphisms, in BRCA1 and/orBRCA2 or a regulator thereof. The detection of variation in BRCA1 andBRCA2 is well-known in the art and is described, for example inEP699754, EP705903, Neuhausen S. L. and Ostrander E. A. Genet. Test(1992) 1, 75-83; Chappnis, P. O. and Foulkes, W. D. Cancer Treat Res(2002) 107, 29-59; Janatova M et al Neoplasma. 2003: 50(4):246-50;Jancarkova N Ceska Gynekol. 2003 68(1): 11-6). Determination ofamplification of the BRCA2 binding factor EMSY is described inHughes-Davies et al Cell 115 523-535).

Mutations and polymorphisms associated with cancer may also be detectedat the protein level by detecting the presence of a variant (i.e. amutant or allelic variant) polypeptide.

A method of identifying a cancer cell in a sample from an individual asdeficient in HR dependent DNA DSB repair may include contacting a samplewith a specific binding member directed against a variant (e.g. amutant) polypeptide component of the pathway, and determining binding ofthe specific binding member to the sample. Binding of the specificbinding member to the sample may be indicative of the presence of thevariant polypeptide component of the HR dependent DNA DSB repair pathwayin a cell within the sample.

Preferred specific binding molecules for use in aspects of the presentinvention include antibodies and fragments or derivatives thereof(‘antibody molecules’).

The reactivities of a binding member such as an antibody on normal andtest samples may be determined by any appropriate means. Tagging withindividual reporter molecules is one possibility. The reporter moleculesmay directly or indirectly generate detectable, and preferablymeasurable, signals. The linkage of reporter molecules may be directlyor indirectly, covalently, e.g. via a peptide bond or non-covalently.Linkage via a peptide bond may be as a result of recombinant expressionof a gene fusion encoding binding molecule (e.g. antibody) and reportermolecule.

The mode of determining binding is not a feature of the presentinvention and those skilled in the art are able to choose a suitablemode according to their preference and general knowledge.

Cancer cells in general are characterised by abnormal proliferationrelative to normal cells and typically form clusters or tumours in anindividual having a cancer condition.

A cancer condition which is deficient in the HR dependent DNA DSB repairpathway as described herein may include any type of solid cancer ormalignant lymphoma and especially leukaemia, sarcomas, skin cancer,bladder cancer, breast cancer, uterus cancer, ovary cancer, prostatecancer, lung cancer, colorectal cancer, cervical cancer, liver cancer,head and neck cancer, oesophageal cancer, pancreas cancer, renal cancer,stomach cancer and cerebral cancer. In some preferred embodiments, thecancer condition may be breast, ovary, pancreas or prostate cancer.Cancers may be familial or sporadic.

A sample obtained from an individual may be a tissue sample comprisingone or more cells, for example a biopsy from a cancerous tissue asdescribed above, or a non-cancerous tissue, for example for use as acontrol.

Methods of the invention may be useful in assessing an individual havinga cancer condition, for example in order to determine a therapeuticcourse of action. A method of assessing an individual having a cancercondition may comprise;

identifying a cancer cell obtained from the individual as deficient inHR dependent DNA DSB repair relative to normal cells, and;

providing a inhibitor of the BER pathway suitable for administration tosaid individual.

In some preferred embodiments, the BER pathway inhibitor is a PARPinhibitor. PARP inhibitors are described in more detail below. A methodof assessing a cancer condition may comprise;

identifying a cancer cell obtained from the individual as deficient inHR dependent DNA DSB repair relative to normal cells, and;

providing a PARP inhibitor suitable for administration to saidindividual.

In some preferred embodiments, the cancer cell which is identified asdeficient in HR dependent DNA DSB repair may have a BRCA1 or BRCA2deficient phenotype.

An individual may have a predisposition to a cancer which is deficientin HR dependent DNA DSB repair. Methods and means of the invention areparticularly useful for such individuals.

An individual may, for example, be heterozygous for a mutation orpolymorphism in a nucleic acid encoding a component of the HR dependentDNA DSB repair pathway, for example a nucleic acid encoding a componentdescribed above.

A method of treatment of cancer in an individual may comprise;administering a BER pathway inhibitor to said individual, wherein saidindividual is heterozygous for a mutation or polymorphism in a geneencoding a component of the HR dependent DNA DSB repair pathway.

A BER inhibitor may be used in the manufacture of a medicament for usein the treatment of a cancer in an individual who is heterozygous for amutation in a gene of a HR dependent DNA DSB repair pathway and a baseexcision repair inhibitor may be used in the treatment of a cancer in anindividual who is heterozygous for a mutation in a gene which encodes acomponent of the HR dependent DNA DSB repair pathway.

In some preferred embodiments, an individual who is heterozygous for amutation or polymorphism in a gene which encodes a component of the HRdependent DNA DSB repair pathway may be heterozygous for a mutation orpolymorphism in BRCA1 and/or BRCA2.

A BER inhibitor suitable for use in a method described herein may be anycompound or entity, such as a small organic molecule, peptide or nucleicacid, which inhibits, reduces or abolishes the activity of one or morecomponents of the BER pathway.

In some preferred embodiments, the BER inhibitor may reduce or abolishthe activity of the enzyme poly (ADP-ribose) polymerase (PARP).

The term PARP as used herein refers to PARP1 (EC 2.4.2.30, Genbank No:M32721) and/or PARP2 (Are et al J. Biol. Chem. (1999) 274 15504-15511;Genbank No: AJ236912) unless context dictates otherwise.

Examples of compounds which are known PARP inhibitors and which may beused in accordance with the invention include:

1. Nicotinamides, such as 5-methyl nicotinamide andO-(2-hydroxy-3-piperidino-propyl)-3-carboxylic acid amidoxime, andanalogues and derivatives thereof.

2. Benzamides, including 3-substituted benzamides such as3-aminobenzamide, 3-hydroxybenzamide. 3-nitrosobenzamide,3-methoxybenzamide and 3-chloroprocainamide, and 4-aminobenzamide, 1,5-di[(3-carbamoylphenyl)aminocarbonyloxy] pentane, and analogues andderivatives thereof.

3. Isoquinolinones and Dihydroisoquinolinones, including2H-isoquinolin-1-ones, 3H-quinazolin-4-ones, 5-substituteddihydroisoquinolinones such as 5-hydroxy dihydroisoquinolinone, 5-methyldihydroisoquinolinone, and 5-hydroxy isoquinolinone, 5-aminoisoquinolin-1-one, 5-dihydroxyisoquinolinone, 3, 4dihydroisoquinolin-1(2H)-ones such as 3, 4dihydro-5-methoxy-isoquinolin-1(2H)-one and 3, 4dihydro-5-methyl-1(2H)isoquinolinone, isoquinolin-1(2H)-ones,4,5-dihydro-imidazo[4,5,1-ij]quinolin-6-ones, 1,6,-naphthyridine-5(6H)-ones, 1,8-naphthalimides such as4-amino-1,8-naphthalimide, isoquinolinone, 3,4-dihydro-5-[4-1(1-piperidinyl) butoxy]-1(2H)-isoquinolinone, 2,3-dihydrobenzo[de]isoquinolin-1-one, 1-11b-dihydro-[2H]benzopyrano[4, 3,2-de]isoquinolin-3-one, and tetracyclic lactams, includingbenzpyranoisoquinolinones such as benzopyrano[4,3,2-de] isoquinolinone,and analogues and derivatives thereof

4. Benzimidazoles and indoles, including benzoxazole-4-carboxamides,benzimidazole-4-carboxamides, such as 2-substituted benzoxazole4-carboxamides and 2-substituted benzimidazole 4-carboxamides such as2-aryl benzimidazole 4-carboxamides and2-cycloalkylbenzimidazole-4-carboxamides including 2-(4-hydroxphenyl)benzimidazole 4-carboxamide, quinoxalinecarboxamides,imidazopyridinecarboxamides, 2-phenylindoles, 2-substitutedbenzoxazoles, such as 2-phenyl benzoxazole and 2-(3-methoxyphenyl)benzoxazole, 2-substituted benzimidazoles, such as 2-phenylbenzimidazole and 2-(3-methoxyphenyl) benzimidazole, 1, 3, 4, 5tetrahydro-azepino[5, 4, 3-cd]indol-6-one, azepinoindoles andazepinoindolones such as 1, 5 dihydro-azepino[4, 5, 6-cd]indolin-6-oneand dihydrodiazapinoindolinone, 3-substituteddihydrodiazapinoindolinones, such as3-(4-trifluoromethylphenyl)-dihydrodiazapinoindolinone,tetrahydrodiazapinoindolinone and 5,6,-dihydroimidazo[4, 5, 1-j, k][1,4]benzodiazopin-7(4H)-one,2-phenyl-5,6-dihydro-imidazo[4,5,1-jk][1,4]benzodiazepin-7(4H)-one and2, 3, dihydro-isoindol-1-one, and analogues and derivatives thereof

5. Phthalazin-1(2H)-ones and quinazolinones, such as4-hydroxyquinazoline, phthalazinone, 5-methoxy-4-methyl-1(2)phthalazinones, 4-substituted phthalazinones,4-(1-piperazinyl)-1(2H)-phthalazinone, tetracyclic benzopyrano[4, 3,2-de]phthalazinones and tetracyclic indeno [1, 2, 3-de] phthalazinonesand 2-substituted quinazolines, such as8-hydroxy-2-methylquinazolin-4-(3H) one, tricyclic phthalazinones and2-aminophthalhydrazide, and analogues and derivatives thereof.

6. Isoindolinones and analogues and derivatives thereof

7. Phenanthridines and phenanthridinones, such as5[H]phenanthridin-6-one, substituted 5[H] phenanthridin-6-ones,especially 2-, 3-substituted 5[H] phenanthridin-6-ones andsulfonamide/carbamide derivatives of 6(5H)phenanthridinones, thieno[2,3-c]isoquinolones such as 9-amino thieno[2, 3-c]isoquinolone and9-hydroxythieno[2, 3-c]isoquinolone, 9-methoxythieno[2,3-c]isoquinolone, and N-(6-oxo-5,6-dihydrophenanthridin-2-yl]-2-(N,N-dimethylamino)acetamide, substituted4,9-dihydrocyclopenta[lmn]phenanthridine-5-ones, and analogues andderivatives thereof.

8. Benzopyrones such as 1, 2-benzopyrone, 6-nitrosobenzopyrone,6-nitroso 1, 2-benzopyrone, and 5-iodo-6-aminobenzopyrone, and analoguesand derivatives thereof.

9. Unsaturated hydroximic acid derivatives such as0-(3-piperidino-2-hydroxy-1-propyl)nicotinic amidoxime, and analoguesand derivatives thereof.

10. Pyridazines, including fused pyridazines and analogues andderivatives thereof.

11. Other compounds such as caffeine, theophylline, and thymidine, andanalogues and derivatives thereof.

Additional PARP inhibitors are described for example in U.S. Pat. Nos.6,635,642, 5,587,384, WO2003080581, WO2003070707, WO2003055865,WO2003057145, WO2003051879, U.S. Pat. No. 6,514,983, WO2003007959, U.S.Pat. No. 6,426,415, WO2003007959, WO2002094790, WO2002068407, U.S. Pat.No. 6,476,048, WO2001090077, WO2001085687, WO2001085686, WO2001079184,WO2001057038, WO2001023390, WO2001021615, WO2001016136, WO2001012199,WO9524379, Banasik et al. J. Biol. Chem., 267:3, 1569-75 (1992), Banasiket al. Molec. Cell. Biochem. 138:185-97 (1994)), Cosi (2002) ExpertOpin. Ther. Patents 12 (7), and Southan & Szabo (2003) Curr Med Chem 10321-340 and references therein.

One preferred class of suitable PARP inhibitors includes phthalazinonessuch as 1(2H)-phthalazinone and derivatives thereof, as described inWO02/36576. In particular, compounds of the formula:

and isomers, salts, solvates, chemically protected forms, and prodrugsthereof, may be used for inhibiting PARP, wherein:

A and B together represent an optionally substituted, fused aromaticring;

R_(c) is represented by -L-R_(L), where L is of formula:

—(CH₂)_(n1)-Q_(2n)-(CH₂)_(n3)—

wherein n₁, n₂ and n₃ are each selected from 0, 1, 2 and 3, the sum ofn₁, n₂ and n₃ is 1, 2 or 3 and Q is selected from O, S, NH, C(═O) or—CR₁R₂—, where R₁ and R₂ are independently selected from hydrogen,halogen or optionally substituted C₁₋₇ alkyl, or may together with thecarbon atom to which they are attached form a C₃₋₇ cyclic alkyl group,which may be saturated (a C₃₋₇cycloalkyl group) or unsaturated (a C₃₋₇cycloalkenyl group), or one of R₁ and R₂ may be attached to an atom inR_(L) to form an unsaturated C₃₋₇ cycloalkenyl group which comprises thecarbon atoms to which R₁ and R₂ are attached in Q, —(CH₂)_(n3)— (ifpresent) and part of R_(L);

and R_(L) is optionally substituted C₆₋₂₀ aryl; and

R_(N) is selected from hydrogen, optionally substituted C₁₋₇ alkyl,C₃₋₂₀ heterocyclyl, and C₅₋₂₀ aryl, hydroxy, ether, nitro, amino, amido,thiol, thioether, sulfoxide and sulfone.

Preferably a compound of the formula:

or an isomer, salt, solvate, chemically protected form, or prodrugthereof may be used to inhibit PARP,

wherein:

A and B together represent an optionally substituted, fused aromaticring;

R_(c) is —CH₂R_(L);

R_(L) is optionally substituted phenyl; and

R_(N) is hydrogen.

In some preferred embodiments, a compound having the structure ofKU-0058684 or KU-0058948 as set out in FIG. 2, or an isomer, salt,solvate, chemically protected form, or prodrug thereof, may be used toinhibit PARP.

Suitable PARP inhibitors are either commercially available or may besynthesized by known methods from starting materials that are known(see, for example, Suto et al. Anticancer Drug Des. 6:107-17 (1991)).

Another class of base excision repair inhibitors includes peptidefragments of components of the BER pathway. For example, peptidefragments of the PARP sequence may be used to inhibit PARP and thusreduce or abolish activity of the BER pathway. Peptide fragments may begenerated wholly or partly by chemical synthesis using the publishedsequences of the components, for example the published PARP sequence(Acc No: NM_001618). Peptide fragments can be readily prepared accordingto well-established, standard liquid or, preferably, solid-phase peptidesynthesis methods, general descriptions of which are broadly available(see, for example, in J. M. Stewart and J. D. Young, Solid Phase PeptideSynthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill. (1984),in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis,Springer Verlag, New York (1984); and Applied Biosystems 430A UsersManual, ABI Inc., Foster City, Calif.), or they may be prepared insolution, by the liquid phase method or by any combination ofsolid-phase, liquid phase and solution chemistry, e.g. by firstcompleting the respective peptide portion and then, if desired andappropriate, after removal of any protecting groups being present, byintroduction of the residue X by reaction of the respective carbonic orsulfonic acid or a reactive derivative thereof.

Other candidate compounds for inhibiting a component of the BER pathway,such as PARP, may be based on modelling the 3-dimensional structure ofthe component and using rational drug design to provide candidatecompounds with particular molecular shape, size and chargecharacteristics. A candidate inhibitor, for example, may be a“functional analogue” of a peptide fragment or other compound whichinhibits the component. A functional analogue has the same functionalactivity as the peptide or other compound in question, i.e. it mayinterfere with the interactions or activity of the DNA repair pathwaycomponent. Examples of such analogues include chemical compounds whichare modelled to resemble the three dimensional structure of thecomponent in an area which contacts another component, and in particularthe arrangement of the key amino acid residues as they appear.

Another class of suitable BER pathway inhibitors includes nucleic acidencoding part or all of the amino acid sequence of a component of theBER pathway, such as PARP (Acc No: NM_001618), or the complementthereof, which inhibit activity or function by down-regulatingproduction of active polypeptide.

For example, the inhibition of PARP activity may be determined usingconventional methods, including for example dot blots (Affar E B et alAnal Biochem. 1998/259(2):280-3), and BER assays that measure the directactivity of PARP to form poly ADP-ribose chains for example by usingradioactive assays with tritiated substrate NAD or specific antibodiesto the polymer chains formed by PARP activity (K. J. Dillon et al,Journal of Biomolecular Screening, 8(3): 347-352 (2003).

For instance, expression of a BER pathway component may be inhibitedusing anti-sense or RNAi technology. The use of these approaches todown-regulate gene expression is now well-established in the art.

Anti-sense oligonucleotides may be designed to hybridise to thecomplementary sequence of nucleic acid, pre-mRNA or mature mRNA,interfering with the production of the base excision repair pathwaycomponent so that its expression is reduced or completely orsubstantially completely prevented. In addition to targeting codingsequence, anti-sense techniques may be used to target control sequencesof a gene, e.g. in the 5′ flanking sequence, whereby the anti-senseoligonucleotides can interfere with expression control sequences. Theconstruction of anti-sense sequences and their use is described forexample in Peyman and Ulman, Chemical Reviews, 90:543-584, (19905 andCrooke, Ann. Rev. Pharmacol. Toxicol. 32:329-376, (1992).

Oligonucleotides may be generated in vitro or ex vivo for administrationor anti-sense RNA may be generated in vivo within cells in whichdown-regulation is desired. Thus, double-stranded DMA may be placedunder the control of a promoter in a “reverse orientation” such thattranscription of the anti-sense strand of the DNA yields RNA which iscomplementary to normal mRNA transcribed from the sense strand of thetarget gene. The complementary anti-sense RNA sequence is thought thento bind with mRNA to form a duplex, inhibiting translation of theendogenous mRNA from the target gene into protein. Whether or not thisis the actual mode of action is still uncertain. However, it isestablished fact that the technique works.

The complete sequence corresponding to the coding sequence in reverseorientation need not be used. For example fragments of sufficient lengthmay be used. It is a routine matter for the person skilled in the art toscreen fragments of various sizes and from various parts of the codingor flanking sequences of a gene to optimise the level of anti-senseinhibition. It may be advantageous to include the initiating methionineATG codon, and perhaps one or more nucleotides upstream of theinitiating codon. A suitable fragment may have about 14-23 nucleotides,e.g. about 15, 16 or 17.

An alternative to anti-sense is to use a copy of all or part of thetarget gene inserted in sense, that is the same, orientation as thetarget gene, to achieve reduction in expression of the target gene byco-suppression; Angell & Baulcombe (1997) The EMBO Journal 16,12:3675-3684; and Voinnet S Baulcombe (1997) Nature 389: pg 553).

Double stranded RNA (dsRNA) has been found to be even more effective ingene silencing than both sense or antisense strands alone (Fire A. et alNature 391, (1998)). dsRNA mediated silencing is gene specific and isoften termed RNA interference (RNAi).

RNA interference is a two-step process. First, dsRNA is cleaved withinthe cell to yield short interfering RNAs (siRNAs) of about 21-23ntlength with 5′ terminal phosphate and 3′ short overhangs (˜2nt). ThesiRNAs target the corresponding mRNA sequence specifically fordestruction (Zamore P. D. Nature Structural Biology, 8, 9, 746-750,(2001)

RNAi may also be efficiently induced using chemically synthesized siRNAduplexes of the same structure with 3′-overhang ends (Zamore P D et alCell, 101, 25-33, (2000)). Synthetic siRNA duplexes have been shown tospecifically suppress expression of endogenous and heterologeous genesin a wide range of mammalian cell lines (Elbashir S M. et al. Nature,411, 494-498, (2001)).

Another possibility is that nucleic acid is used which on transcriptionproduces a ribozyme, able to cut nucleic acid at a specific site—thusalso useful in influencing gene expression. Background references forribozymes include Kashani-Sabet and Scanlon, 1995, Cancer Gene Therapy,2(3): 213-223, and Mercola and Cohen, 1995, Cancer Gene Therapy, 2(1),47-59.

Methods of the invention may comprise administering a BER inhibitor,such as a PARP inhibitor, to an individual. This may occur subsequent tohaving identified the individual as having a cancer condition deficientin HR dependent DMA DSB repair.

While it is possible for the active compound to be administered alone,it is preferable to present it as a pharmaceutical composition (e.g.,formulation) comprising at least one active compound, as defined above,together with one or more pharmaceutically acceptable carriers,adjuvants, excipients, diluents, fillers, buffers, stabilisers,preservatives, lubricants, or other materials well known to thoseskilled in the art and optionally other therapeutic or prophylacticagents.

Pharmaceutical compositions comprising Base excision repair inhibitorsas defined above, for example an inhibitor admixed together with one ormore pharmaceutically acceptable carriers, excipients, buffers,adjuvants, stabilisers, or other materials, as described herein, may beused in the methods described herein.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of a subject (e.g., human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation.

Suitable carriers, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Suchmethods include the step of bringing the active compound intoassociation with a carrier which may constitute one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

Formulations may be in the form of liquids, solutions, suspensions,emulsions, elixirs, syrups, tablets, lozenges, granules, powders,capsules, cachets, pills, ampoules, suppositories, pessaries, ointments,gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses,electuaries, or aerosols.

The BER inhibitor or pharmaceutical composition comprising the inhibitormay be administered to a subject by any convenient route ofadministration, whether systemically/peripherally or at the site ofdesired action, including but not limited to, oral (e.g. by ingestion);topical (including e.g. transdermal, intranasal, ocular, buccal, andsublingual); pulmonary (e.g. by inhalation or insufflation therapyusing, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal;parenteral, for example, by injection, including subcutaneous,intradermal, intramuscular, intravenous, intraarterial, intracardiac,intrathecal, intraspinal, intracapsular, subcapsular, intraorbital,intraperitoneal, intratracheal, subcuticular, intraarticular,subarachnoid, and intrasternal; by implant of a depot, for example,subcutaneously or intramuscularly.

Formulations suitable for oral administration (e.g., by ingestion) maybe presented as discrete units such as capsules, cachets or tablets,each containing a predetermined amount of the active compound; as apowder or granules; as a solution or suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion; as a bolus; as an electuary; or as apaste.

A tablet may be made by conventional means, e.g., compression ormolding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing in a suitable machine the activecompound in a free-flowing form such as a powder or granules, optionallymixed with one or more binders (e.g., povidone, gelatin, acacia,sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers ordiluents (e.g., lactose, microcrystalline cellulose, calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, silica);disintegrants (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose); surface-active ordispersing or wetting agents (e.g., sodium lauryl sulfate); andpreservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,sorbic acid). Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activecompound therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile. Tablets mayoptionally be provided with an enteric coating, to provide release inparts of the gut other than the stomach.

Formulations suitable for parenteral administration (e.g., by injection,including cutaneous, subcutaneous, intramuscular, intravenous andintradermal), include aqueous and non-aqueous isotonic, pyrogen-free,sterile injection solutions which may contain anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. Examples of suitable isotonic vehicles for use insuch formulations include Sodium Chloride Injection, Ringer=s Solution,or Lactated Ringer's Injection. Typically, the concentration of theactive compound in the solution is from about 1 ng/ml to about 10 μg/ml,for example from about 10 ng/ml to about 1 fig/ml. The formulations maybe presented in unit-dose or multi-dose sealed containers, for example,ampoules and vials, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets. Formulations may be in the form ofliposomes or other microparticulate systems which are designed to targetthe active compound to blood components or one or more organs.

It will be appreciated that appropriate dosages of the active compounds,and compositions comprising the active compounds, can vary from patientto patient. Determining the optimal dosage will generally involve thebalancing of the level of therapeutic benefit against any risk ordeleterious side effects of the treatments of the present invention. Theselected dosage level will depend on a variety of factors including, butnot limited to, the activity of the particular compound, the route ofadministration, the time of administration, the rate of excretion of thecompound, the duration of the treatment, other drugs, compounds, and/ormaterials used in combination, and the age, sex, weight, condition,general health, and prior medical history of the patient. The amount ofcompound and route of administration will ultimately be at thediscretion of the physician, although generally the dosage will be toachieve local concentrations at the site of action which achieve thedesired effect without causing substantial harmful or deleteriousside-effects.

Compositions comprising BER pathway inhibitors may be used in themethods described herein in combination with standard chemotherapeuticregimes that damage cancer cell DNA. Suitable agents may includeinhibitors of topoisomerase I and II activity, such as camptothecin,drugs such as irinotecan, topotecan and rubitecan, alkylating agentssuch as temozolomide and DTIC (dacarbazine), and platinum agents likecisplatin, cisplatin-doxorubicin-cyclophosphamide, carboplatin, andcarboplatin-paclitaxel.

Other suitable chemotherapeutic agents includedoxorubicin-cyclophosphamide, capecitabine,cyclophosphamide-methotrexate-5-fluorouracil, docetaxel,5-flouracil-epirubicin-cyclophosphamide, paclitaxel, vinorelbine,etoposide, pegylated liposomal doxorubicin and topotecan.

The treatment of individuals using such agents is well-known in the art.

Administration in vivo can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician.

In general, a suitable dose of the active compound is in the range ofabout 100 μg to about 250 mg per kilogram body weight of the subject perday. Where the active compound is a salt, an ester, prodrug, or thelike, the amount administered is calculated on the basis of the parentcompound and so the actual weight to be used is increasedproportionately.

Methods of the invention may also be useful in investigating andassessing a cancer condition in an individual.

A method of assessing the activity of the HR dependent DNA DSB repairpathway in a cancer condition may comprise;

contacting a base excision repair inhibitor with a sample of cancercells obtained from the individual having the condition, and;

determining the amount of cell death in said sample relative to acontrol sample.

An increase in cell death in the sample relative to control cells whichhave normal levels of HR dependent DNA DSB repair activity is indicativethat the cancer is deficient in HR dependent DNA DSB repair.

The individual may have a cancer condition and the sample may be asample of cancer cells, for example from a tumour biopsy.

In preferred embodiments, the base excision repair inhibitor is a PARPinhibitor. A method of assessing HR dependent DNA DSB repair in a cancercondition may thus comprise;

contacting a PARP inhibitor with a sample of cancer cells obtained fromthe individual having the cancer condition, and;

determining the amount of cell death in said sample relative to acontrol sample.

An increased sensitivity the PARP inhibitor in cells from the samplerelative to control cells is indicative that the cancer is deficient inthe HR dependent DNA DSB repair activity.

Increased sensitivity to PARP inhibitors may be indicative that thecancer cells have a BRCA1 or BRCA2 deficient phenotype, for example areduction or abolition of BRCA1 or BRCA2 expression or activity.

A cancer condition identified as being deficient in HR dependent DNA DSBrepair activity, for example a condition having a BRCA1 or BRCA2deficient phenotype, may be subjected to therapies that are specificallydirected at such conditions. Suitable therapies may include the use ofDNA cross-linking agents such as Mitomycin C, cisplatin or carboplatin.

Methods may be used to predict the response of a cancer condition in anindividual to a treatment targeting HR, for example a treatment specificto cancers having a BRCA1 or BRCA2 deficient phenotype.

A method of predicting the response of a cancer condition in anindividual to a treatment targeting cancers deficient in HR dependentDNA DSB repair may comprise;

contacting a BER inhibitor, for example a PARP inhibitor, with a sampleof cancer cells obtained from the individual having the cancercondition, and;

determining the amount of cell death in said sample relative to acontrol sample.

An increase in cell death in the sample relative to control cells whichhave normal levels of HR dependent DMA DSB repair activity (i.e. anincreased sensitivity to PARP inhibitors) is indicative that the cancermay be responsive to said treatment.

Treatments which target cancers deficient in HR dependent DNA DSBrepair, for example BRCA1 or BRCA2 deficient cancers, may include, forexample, DNA cross-linking agents such as mitomycin C, cisplatin orcarboplatin.

Other aspects of the invention relate to the use of an inhibitor of HRdependent DNA DSB repair in the treatment of a cancer that is deficientin base excision repair.

A method of treatment of a cancer deficient in base excision repair inan individual may comprise;

administering an HR dependent DNA DSB repair pathway inhibitor to saidindividual.

An HR dependent DNA DSB repair inhibitor may be used in the manufactureof a medicament for use in the treatment of cancer in an individual,wherein said cancer is deficient in base excision repair.

An inhibitor of HR dependent DNA DSB repair may include an inhibitor ofone or more of the pathway components set out above. Suitable inhibitorsinclude ATM inhibitors.

An ATM inhibitor may, for example, be a compound of formula I:

or an isomer, salt, solvate, chemically protected form, or prodrugthereof, wherein:

Y is either 0 or S;

R¹ and R² are independently hydrogen, an optionally substituted C₁₋₇alkyl group, C₄₋₂₀ heterocyclyl group, or C₅₋₂₀ aryl group, or maytogether form, along with the nitrogen atom to which they are attached,an optionally substituted heterocyclic ring having from 4 to 8 ringatoms; and

R³ is a phenyl group attached to an optionally substituted C₅₋₂₀carboaryl group by an ether or thioether bridge, the phenyl group andoptionally substituted C₅₋₂₀ carboaryl group being optionally linked bya further bridge group, which is bound adjacent the ether or thioetherbridge on both groups so as to form an optionally substituted C₅₋₂₀oxygen or sulphur containing heterocycle fused to both the phenyl groupand the C₅₋₂₀ carboaryl group, the phenyl group being further optionallysubstituted.

Such inhibitors are described in more detail in WO03/070726.

Other inhibitors include peptidyl fragments of components of HRdependent DNA DSB repair and encoding nucleic acids as described above.

A cancer condition may be identified as deficient in BER activity usinga method described above.

Aspects of the present invention will now be illustrated with referenceto the accompanying figures described below and experimentalexemplification, by way of example and not limitation.

Further aspects and embodiments will be apparent to those of ordinaryskill in the art.

Various parameters and features of the invention are set out above. Forthe avoidance of doubt, it is stated that all combinations andsub-combinations of these parameters and features are encompassed by thepresent invention.

All documents mentioned in this specification are hereby incorporatedherein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings.

It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1 shows that a reduction in the level of Parp1 reduces theviability of BRCA1 and BRCA2 mutant cells relative to wild-type cells.

FIG. 2 shows the PARP inhibitors KU0058684, KU0058948 and KU0051529 andtheir IC₅₀s against PARP-1 enzyme activity.

FIGS. 3 and 4 show clonogenic survival curves of cells exposed to PARPinhibitors.

FIG. 3 shows Brca1 wild type (11CO:▪), heterozygous (Cre6:▴) anddeficient (Cre10:●) ES cells under continuous exposure to PARPinhibitors (KU0058684, top; KU0058948, middle; KU0051529, bottom). Errorbars represent standard errors of the mean.

FIG. 4 shows Brca2 wild type (D3:▪), heterozygous (Cre6:▴) and deficient(Cre24:●) ES cells under continuous exposure to PARP inhibitors(KU0058684, top; KU0058948, middle; KU0051529, bottom). Error barsrepresent standard errors of the mean.

FIGS. 5 and 6 show clonogenic survival curves after 1, 4 and 24 hourtimed exposures to KU0058684.

FIG. 5 shows Brca1 wild type (11CO:▪), heterozygous (Cre6:▴) anddeficient (Cre10:●) ES cells after 1 (left), 4 (middle) and 24 hour(right) timed exposures to KU0058684. Error bars represent standarderrors of the mean.

FIG. 6 shows Brca2 wild type (D3:▪), heterozygous (Cre6:▴) and deficient(Cre24:●) ES cells after 1 (left), 4 (middle) and 24 hour (right) timedexposures to KU0058684. Error bars represent standard errors of themean.

FIGS. 7 and 8 show that PARP inhibition in BRCA-1 and BRCA-2 mutantcells treated with PARP inhibitor resulted in enhanced G2/M arrest.

FIG. 7 shows Brca1 wild type (11CO:top) and mutant (Cre10:bottom) cellstreated with KU0058684 for 24 h at 0 nM (left), 10 nM (middle) or 1 μM(right) and analysed by FACS.

FIG. 8 shows Brca2 wild type (D3) and mutant (Cre24) cells treated withKU0058684 for 24 h at 0 nM (left), 10 nM (middle) or 1 μM (right) andanalysed by FACS.

FIG. 9 shows a quantitative analysis of Rad51 foci formation induced byPARP inhibition in wild-type cells but not in Brca1 or Brca2 defectivecells.

FIG. 10 shows a neutral comet analysis of BRCA2−/− VC8 and BRCA2complemented VC8-BAC. KU0058684 (1 μM) treatment for 30 hours induces asignificant increase in DNA DSBs as judged by an increase in tail momentin the BRCA2−/− cells whilst no significant increase in tail moment isobserved in the BRCA2 complemented line. Average data from 3 independentexperiments is shown+/−SEM, with 50 comets being scored for tail momentin each experiment.

FIG. 11A-E shows a possible model for the selective effects of PARPinhibition on BRCA1 and BRCA2 mutant cells.

FIGS. 12 and 13 show phospho-histone H3 FACS data for ES cells FIG. 12shows phospho-histone H3 FACS data for Brca1 wild type (11CO:top) andmutant (Cre10:) ES cells and Brca2 wild type (D3) and mutant (Cre24)cells treated with KU0058684 for 24 h at 0 nM (left), 10 nM (middle) or1 μM (right) and analysed by FACS.

FIG. 13 shows phospho-histone H3 FACS for VC8 and VC8BAC cells treatedwith 0 μM, 100 μM, 1 nM and 10 nM (left to right, respectively)KU0058684 for 24 h.

FIGS. 14 and 15 show an analysis of the effects of PARP inhibition inother cells lacking BRCA1 and BRCA2 function.

FIG. 14 shows clonogenic survival curves of Brca2 deficient (V-C8:▪) andcomplemented (V-C8 BAC+:▴) cells under continuous exposure to PARPinhibitors (KU0058684: top, KU0058948: middle and KU0051529: bottom).

FIG. 15 shows clonogenic survival curves of Brca2 deficient (V-C8:▪) andcomplemented (V-C8 BAC+:▴) cells after 1 hour (top), 4 hour (middle) and24 hour (bottom) timed exposures to KU0058684. Error bars representstandard errors of the mean.

FIG. 16 shows tumour formation in ES xenografts and the effect oftreatment with KU0058684; dotted line—wild type with vehicle, bold solidline—wild type with drug KU0058684, solid line—Brca 2 deficient withvehicle, dashed line—Brca2 deficient with KU0058684.

FIG. 17 shows clonal survival curves of BRCA1 wild-type (MCF7-scrambled)and BRCA1 silenced (MCF7-3.23) cells under continuous exposure to arange of concentrations of the PARP inhibitor KU0058684 for 12-14 days.Log concentration of inhibitor is plotted against log surviving fractionof cells. Error bars represent standard errors of the mean.

FIG. 18 shows clonal survival curves of BRCA1 wild-type (MCF7-scrambled)and BRCA1 silenced (MCF7-3.23) cells under continuous exposure to arange of concentrations of PARP inhibitor KU0051529 for 12-14 days. Logconcentration of inhibitor is plotted against log surviving fraction ofcells. Error bars represent standard errors of the mean.

EXAMPLES

Materials and Methods

RNA Interference

Gene-specific pSUPER (T. R. Brunmelkamp et al Science 296, 550-3 (2002))constructs were generated expressing the following RNAi targetsequences: (i) mouse Parp1 5′-GCGGAGUACGCCAAGUCCA-3′ (SEQ ID NO:1) (ii)scrambled control 5′-CAUGCCUGAUCCGCUAGUC-3′(SEQ ID NO:2).

A 1.6 kb fragment containing the CMV IE promoter and eCFP (enhanced cyanfluorescent protein) was subcloned from pECFP-Mito (Invitrogen) into theSapI site of the resultant pSUPER constructs, generatingpSUPER-eCFP-Parp1 and pSUPER-eCFP-control. D3 ES cells were transfectedwith these plasmids using Lipofectamine 2000 (Invitrogen) according tothe manufacturers instructions. Forty-eight hours after transfection,total cell lysates were generated using a buffer composed of 20 mM TrispH 8, 200 mM NaCl, 1 mM EDTA, 0.5% (v/v) NP40, 10% (v/v) glycerol andprotease inhibitors. 30 μg of each lysate was electrophoresed onBis-Tris Acetate Acrylamide Pre Cast Gels (Novex) and blotted ontoTrans-Blot Nitrocellulose (Biorad). Blots were probed with either Rabbitpolyclonal anti-PARP-1 antibody (Cell Signalling, Cat No. 9542) orRabbit anti-GFP/CFP antiserum (Invitrogen, Cat. No. R970-01), followedby a secondary hybridization with anti-rabbit IgG-HRP with subsequentchemiluminescent detection using ECL™ (Amersham, UK).

Small Molecule Inhibitors of PARP:

PARP inhibitors were synthesized as described in WO02/36576. Chemicalinhibitors were dissolved in DMSO at 10 mM and stored at −20° C. in thedark.

Cell Lines

VC8 cells and the mouse Brca2 BAC complemented derivatives were asdescribed in M. Kraakman-van der Zwet et al., Mol Cell Biol 22, 669-79(2002)). ES cells defective in Brca2 function have been describedpreviously (Tutt et al. (2002) EMBO Rep 3, 255-60). The construction ofES cells defective in Brca1 will be described elsewhere but havepreviously been validated (Foray et al. (2003) Embo J 22 2860-71).HBL100 cells were transfected with a pSUPER BRCA1 RNAi plasmid andselected with geneticin for 3 weeks. Clones were selected on the basisof their BRCA1 expression, as analysed by Northern blot.

Clonogenic Assays For measurement of sensitivity to Parp1 RNA knockdown,ES cells maintained on tissue culture dishes coated with 0.1% gelatinwere transfected as above, with either pSUPER-eCFP-Parp1 orpSUPER-eCFP-control along with a vector expressing resistance to theantibiotic, blasticidin (pEF-Bsd, Invitrogen). Twenty-four hours aftertransfection, cells were trypsinized and seeded in 6-well plates.

Forty-eight hours post-transfection, treatment with blasticidin wascommenced and cells were re-fed every three days. After 10-14 days,cells were washed with PBS, fixed in methanol and stained with crystalviolet. Colonies containing greater than approximately 50 cells werecounted.

For measurement of sensitivity to chemical inhibitors, cell cultures inexponential growth were trypsinised and seeded at various densities in6-well plates onto Mitomycin C inactivated mouse embryonic fibroblastsand where appropriate treated with inhibitors after 18 h. For continuousexposure, cells were re-fed every 4 days with fresh medium andinhibitor. For timed exposures, inhibitor was added for the specifiedperiod then cells were washed and re-fed with fresh medium. After 10-14days, cells were washed with PBS, fixed in methanol and stained withcrystal violet. Colonies containing greater than approximately 50 cellswere counted. Experiments were performed at least three times intriplicate.

FACS Analysis

For DNA content measurement, cells were fixed in 703 ethanol, incubatedwith Rnase A and propidium iodide (PI) and analysed with a FACSCalibur(Becton Dickinson). For phospho-Histone H3 analysis, cells were fixed in70% ethanol, permeabilised with 0.253 triton X-100, incubated withanti-phospho-histone H3 antibody (Upstate Biotechnology) for 3 hours,and then with FITC-anti rabbit IgG (Serotec) for 30 minutes. Processingfor FACS analysis was as above.

Apoptotic Analysis

Cells were trypsinised, retaining both culture supernatant and washmedium. These were pooled and the cells washed in cold PBS-A beforeresuspension at 1×10⁶ cells/ml in binding buffer (10 mM HEPES, 140 mMNaCl, 2.5 mM CaCl₂ (pH7.4)). 100 μl suspension was incubated in the darkwith 5 μl Annexin V-FITC (BD Biosciences)/0.1 μg propidium iodide for 15mins at room temperature, 400 μl binding buffer added and analysedimmediately on a FACS Calibur (BD Biosciences).

Rad 51 Focus Formation

ES cells were cultured for 48 h in various concentrations of PARPInhibitor fixed in 4% paraformaldehyde in PBS and permeabilised with0.23 Triton X100 In PBS. Cells were stained with a 1:100 dilution ofrabbit anti-Rad51 ployclonal antibody (Ab 551922, BD-Pharmingen, Oxford,UK). After washing, the primary antibody was visualised with AlexaFluor-555 goat anti-rabbit IgG (Alexa) and nuclei with TO-PRO-3 iodide(Molecular Probes). Rad51 foci were visualised with and quantified usinga Leica TCS-SP2 confocal microscope.

Comet Assay

VC8 and VC8-BAC cells were plated 24 hours prior to treatment with 1 μMKU0058684 for 30 hours. All further work was carried out in the dark.Cells were washed with and scraped into PBS prior to Comet analysis asdescribed (Lemay and Wood, 1999). Cells suspended in LMP agarose (0.53in PBS) were spread onto Comet slides (Trevigen, Gaithersburg) andplaced at 4 C until set, prior to lysis for 45 mins in 2.5M NaCl, 100 mMEDTA, 10 mM Tris Base, 13 sodium lauryl sarcosinate, 0.01% Triton X-100.Slides were transferred to TBE for 5 mins prior to electrophoresis at18V for 15 mins. Slides were then fixed in 1003 ethanol for 5 mins andair dried before the addition of SYBR green dye and visualisation byepifluorescence using fluorescien filters (Nikon). Comets were analysedusing the Comet software module of the Lucia G imaging package assupplied by Nikon. 50 comets per data point were examined for each ofthree independent experiments and the mean tail moment calculated.

Mitotic Chromosome Analysis

ES cells were seeded onto gelatin, treated for 24 hours with chemicalinhibitors, followed by colcemid treatment for 1 hour. Cells wereharvested, fixed, dropped onto slides, dried and stained with DAPIbefore chromosome analysis under a microscope.

ES Cell Xenografts and Treatment with KU0058684

ES cell derived tumours (teratomas) were produced by subcutaneousinjection of 2×10⁸ ES cells into 6-8 week athymic BALB/c nude (nu/nu)mice. Twenty mice were injected with Brca2 deficient ES cells and anidentical cohort with isogenic wild type cells. Two days after cellinjection, treatment with KU0058684 or vehicle was initiated. For threeconsecutive days, two intraperitoneal doses of KU0058684 (or vehicle)were administered, six hour apart, each at a dosage of 15 mg/kg animal.This treatment was then stopped for five days and then reinitiated (asbefore) for another three consecutive days. Growth of tumours wasmonitored from a minimum volume of 0.3 cm³. The data in FIG. 16represents two separate experiments involving in total 40 animals.

Production of BRCA1 Cell-Deficient Line

The MCF7 scrambled and MCF7-3.23 cell lines were generated by stabletransfection of MCF7 mammary adenocarcinoma cells with gene specificpSUPER constructs. Gene specific pSUPER constructs were generatedexpressing the following RNAi target sequences: (i) human BRCA15′-GGAACCTGTCTCCACAAAG-3′ (SEQ ID NO:3) (ii) scrambled control5′-CATGCCTGATCCGCTAGTC-3′(SEQ ID NO:4). A 1.8 kb fragment containing thehuman EF1a promoter and the blasticidin resistance gene (bsd) wassubcloned from pEFBsd (Invitrogen) into the SapI site of the resultantpSUPER constructs, generated pSUPER-Bsd-BRCA1 and pSUPER-Bsd-scrambled.MCF7 cells were transfected with these plasmids using FuGene6 (Roche)according to the manufacturers instructions. After selection inblasticidin, resistant clones were assessed by real time PCR forsilencing of BRCA1 mRNA (Egawa et al Oncology. 2001; 61(4): 293-8; Egawaet al Int J Cancer. 2001 Jul. 20; 95(4):255-9). Clones with reducedlevels of BRCA1 mRNA were cultured (under blasticidin selection) over 8passages and the real-time assay repeated. Cell line MCF7-3.23 was shownto have only 30% expression of BRCA1 compared to MCF7 clones possessingthe construct pSUPER-Bsd-scrambled.

Results

Reduction of Parp1 Protein Levels by siRNA

A plasmid (pSUPER-eCFP-Parp1) expressing a Parp1 specific siRNA underthe control of the HI promoter (T. R. Brununelkamp et al, Science 296,550-3 (2002)) and Enhanced Cyan Fluorescent Protein (eCFP) under thecontrol of the CMV IE promoter was transfected into D3 mouse embryonicstem cells. As a control, a plasmid expressing an unrelated scrambledsiRNA, pSUPER-eCFP-control was separately transfected. Forty-eight hoursafter transfection, cell lysates were prepared and analysed by westernblotting. Blots were probed with either a polyclonal anti-PARP-1antibody or an anti-GFP/CFP antiserum.

Levels of PARP1 in the Parp1 specific siRNA expressing cells wereobserved to be much lower than PARP1 levels in the control cells. Levelsof eCFP were similar in both Parp1 siRNA expressing and control cells.

Reduction in the viability of BRCA1 and BRCA2 deficient cells afterParp1-specific siRNA knockdown.

Mild type, Brca1^(−/−) and Brca2^(−/−) mouse embryonic stem (ES) cellswere transfected with either pSUPER-eCFP-Parp1 or pSUPER-eCFP-controltogether with a blasticidin resistance-encoding plasmid pEF-Bsd in a10:1 ratio. Blasticidin resistance clones were selected and quantitated.The results are shown in FIG. 1 plotted as the number of colonies aftertransfection of pSUPER-eCFP-Parp1 relative to the number aftertransfection of pSUPER-eCFP-control. Error bars are equal to onestandard deviation around the mean.

After correction for transfection efficiency using the control siRNA, itwas apparent that the survival of both Brca1 and Brca2 deficient EScells was considerably reduced when the expression of Parp1 wasinhibited.

Reduction in the Viability of BRCA1 and BRCA2 Deficient Cells afterChemical PARP Inhibitors

Chemical inhibitors of Parp activity were employed to confirm theselective inhibition of Brca1 and Brca2 deficient cells observed above.Two different PARP inhibitors, KU0058684, KU0058948 and a weakly activebut chemically related compound KU0051529 were used (FIG. 2). Thesenovel PARP inhibitors are based around a phthalazin-1-one core and arecompetitive inhibitors with respect to the PARP substrate NAD⁺.KU0058684 and KU0058948 are potent and specific inhibitors of thepoly(ADP-ribose) polymerase activity of the proteins PARP-1 and PARP-2and do not inhibit vault PARP, tankyrase or PARP-3 at concentrations upto 1 μM. Conversely, KU0051529 is ˜250× less effective in the inhibitionof these enzymes despite being chemically related.

KU0058684, KU0058948 and KU0051529 were used to probe the sensitivity ofcells deficient in Brca1 or Brca2 to the inhibition of PARP activity.Clonogenic assays showed that both Brca1 and Brca2 deficient cell lineswere extremely sensitive to KU0058684 and KU0058948 compared tootherwise isogenic cells (FIG. 3, 4). The SF50 (dosage at which 50% ofcells survived) for KU0058684 was 3.5×10⁻⁸ M for Brca1 and 1.5×10⁻⁸ Mfor Brca2; for wild-type cells this was around 3.5×10⁻⁸ M. Thisrepresents factors of 57-fold and 133-fold enhanced sensitivity forBrca1 and Brca2 mutant cells respectively compared to wild-type. Similarresults were obtained with Chinese hamster ovary cells deficient inBrca2, which showed a greater than 1000-fold enhanced sensitivitycompared to a Brca2-complemented derivative (FIGS. 14 and 15). Thesensitivity of Brca1 and Brca2 mutant cells to KU0058948 was evengreater than that of KU0058684. In contrast, KU0051529 had no selectiveeffect on cells lacking wild-type Brca1 or Brca2 compared to wild-typecells. This, in conjunction with the siRNA data, demonstrates that themechanism of sensitivity is specifically through inhibition of PARP.Notably none of the inhibitors had any selective effect on cellsheterozygous for Brca1 or Brca2 mutation.

Time course dependence of the effects on KU0058684 on clonogenicsurvival of Brca1 and Brca2 deficient cells Cells were exposed todifferent concentrations of KU0058684 for defined periods of time. Theinhibitor was then removed and the effects measured using a clonogenicassay. The inhibitory effects of KU0058684 on clonal growth wereapparent after a relatively short exposure time, 4 h, and wereessentially complete by 24 h exposure (FIGS. 5 and 6). The effects ofPARP inhibition were found to be irreversible as a short exposurefollowed by 10-14 days in the absence of the inhibitor prevents growth.

Effect of PARP Inhibition on Cell Cycle Arrest

FACS analysis was used to determine whether PARP inhibition resulted incell cycle arrest. Cells were exposed with KU0058684 for various periodsthen labelled with BrdU and the proportion of cells in each phase of thecell cycle. The results are shown in FIGS. 1 and 8. KU0058684 wasobserved to elicit a profound arrest of cells with a tetraploid DNAcontent indicating arrest in G₂ or M phase of the cell cycle. To furthercharacterise this arrest, cells were analysed for both DNA content andfor phosphor yl a ted Histone H3, an M phase marker. To furthercharacterise this arrest, cells were analysed for both DNA content andfor phosphorylated Histone H3, an M phase marker (FIGS. 12 and 13). Themajority of arrested cells did not label with anti-phospho histone H3antibodies indicating that the majority of cells were arrested in G2.

Rad51 Foci Formation

One hallmark of Brca-dependent double-strand break repair is theformation of foci in the nucleus containing Rad51. The ability ofKU0058684 to elicit Rad51 foci in wild-type and in Brca1 and Brca2deficient cells was investigated.

Wild-type, and Brca1 and Brca2 defective ES cells were exposed todiffering concentrations of KU0058684 for 48 hours. Cells were thenfixed and stained for RAD51 foci as described by Tarsounas (Tarsounas Met al Oncogene. 2003 22(8):1115-23).

In wild-type ES cells, KU0058684 caused Rad51 foci formation in adose-dependent fashion (FIG. 9). In contrast, no foci were induced inBrca1 or Brca2 deficient cells. This latter finding is consistent withprevious observations that DNA damaging agents cannot cause Rad51 focusformation in Brca1 or Brca2 deficient cells.

KU0058684 is therefore shown to induce lesions, such as double-strandDNA breaks or lesions that degenerate into double-strand DNA breaks,which are repaired by a complex that involves Rad51 and which requiresBrca1 and Brca2. Importantly, KU0051529 did not induce Rad51 focusformation at comparable doses, emphasizing the specificity of mechanismof sensitisation.

Comet Assays

To determine whether inhibition of PARP activity leads to the productionof DNA double-strand breaks, neutral comet assays were performed onBrca2 mutant cells and their isogenic counterparts. The results areshown in FIG. 10. After a 30 hour exposure to 1 μM KU0058684, there wasa 4.7 fold increase in the tail moment of the Brca2 deficient VC8 cellsand no significant increase in tail moment for the complemented VC8-BACline. This result shows that DNA double strand breaks induced by thePARPi are left unrepaired in the Brca2 deficient line.

Mitotic Chromosome Analysis

Examination of mitotic chromosomes of Brca1 and Brca2 deficient cellsrevealed that KU0058684 treatment resulted in frequent majoraberrations. These included chromatid breaks and tri-radial andquadri-radial chromosomes. These phenotypes provide indication of afailure to repair double-strand breaks by sister chromatid geneconversion and the elevated use of alternative error-prone pathways.

ES Cell Xenografts and Treatment with KU0058684

Isogenic and Brca2 deficient ES cell derived tumours (teratomas) wereproduced in athymic BALB/c nude (nu/nu) mice as described above.

The effect of KU0058684 on the growth of wild-type and Brca2 deficientxengraft tumours was measured and the results shown in FIG. 16.

KU0058684 was observed to dramatically reduce the growth of Brca2deficient tumours relative to wild-type tumours.

Effect of PARP Inhibition in BRCA1 Cell-Deficient Lines

The MCF7-scrambled and MCF7-3.23 cell lines were produced as describedabove. MCF7-3.23 was found to have 30% expression of BRCA1 compared toMCF7-scrambled.

Both MCF7-scrambled and MCF7-3.23 cells were treated with KU0058684 andKU0051529 and the survival of cells determined (FIGS. 17 and 18).KU0058684 is a potent PARP inhibitor whereas KU0051529 is lesseffective.

Neither MCF7-scrambled nor MCF7-3.23 cells displayed significantsensitivity to KU0051529 (FIG. 18). However both cell lines weresensitive to KU0058684.

MCF7-3.23 cells were shown to be significantly more sensitive toKU0058684 than MCF7-scrambled cells.

Interaction of PARP with the HR dependent DNA DSB pathway Withoutlimiting the scope of the invention in any way, one possible model forinteraction between PARP and the HR dependent DNA DSB pathway is shownin FIG. 11.

DNA single-strand breaks (SSB) form due to oxidative damage and itsrepair. Inhibition of Parp-1 PAR polymerase activity prevents therecruitment of the XRCC1 scaffold protein and subsequent SSB gap fillingby DNA polymerises (FIG. 11A).

Large numbers of SSBs persist and are encountered by DNA replicationforks. The absence of a template strand at the SSB leads to a DSB andmay, dependent on position, cause replication fork collapse (FIG. 11B).

The proximity of an undamaged sister chromatid template allows theinvasion of the sister chromatid by RAD51 coated single stranded DNAfilament and initiation of sister chromatid recombination repair.

This process is dependent on BRCA1 and BRCA2 and is associated with theformation of multiple nuclear foci of RAD51. Collapsed replication forkscan be restarted by a similar mechanism (FIG. 11C).

When Holliday junctions at recombination intermediates are resolved, asister chromatid exchange (SCE) may occur. The excess number of SSBsencountered by replication forks during Parp-1 inhibition leads to anincrease in SCEs (FIG. 11D).

In the absence of functional BRCA1 or BRCA2, RAD51 focus formation andsister chromatid recombination are severely impaired. The excessunrepaired SSBs form DNA DSBs during DNA replication but sisterchromatid recombination does not occur. They remain unrepaired aschromatid breaks or are repaired by error prone RAD51 independentmechanisms causing complex chromosome rearrangements. These cells arrestwhen they encounter the G2/M DNA damage checkpoint and permanentlyarrest or apoptose (FIG. 11E).

RNAi data provided herein, along with the observation that KU0051529 wasineffective, demonstrate that PARP inhibition is responsible for thesensitisation effects observed.

PARP inhibition induces lesions that are normally repaired by sisterchromatid exchange (SCE) (Hang Z Q, et al (1997) Genes Dev. Vol. 11(18):2347-58 and ‘From DNA damage and stress signalling to cell death’G. de Murcia and S. Shall eds. Oxford University Press (2000)). PARPinhibition is known to increase SCE, with no concomitant increase ingene conversion of DSBs and therefore no global increase in theRad51-dependent recombination pathway (Schultz N et al 2003, NucleicAcids Research; vol. 31 (17): 4959-4964).

The synthetic lethality approach described herein may be useful both inthe treatment of a) in tumours in BRCA carriers b) in tumours whereother components of HR dependent DNA DSB repair are defective.

Although differences have been described in the age of onset andpathology of tumours in carriers of BRCA mutations, treatment is thesame at present as for patients with sporadic disease. The presentinvention provides a new approach for these tumours is described herein.

Significantly, no heterozygous effect was observed. This is importantfor the therapeutic use of the described methods in heterozygouscarriers of mutations in the HR dependent DNA DSB repair pathway,including, for example BRCA1/2 mutations.

1. A method of determining whether an individual having a cancercondition will respond to PARP inhibitor treatment comprising; providinga sample of cancer cells obtained from an individual having a cancercondition, and identifying one or more cancer cells in the sample asdeficient in HR dependent DNA DSB repair relative to normal cells,wherein the presence of one or more cancer cells deficient in HRdependent DNA DSB repair indicates that the individual will respond tosaid treatment.
 2. A method according to claim 1 comprising providing aPARP inhibitor suitable for administration to said individual.
 3. Amethod according to claim 1 comprising administering a PARP inhibitor tosaid individual.
 4. A method according to claim 1 wherein said cancercells are identified as deficient in a HR dependent DNA DSB repairpathway by determining the HR dependent DNA DSB repair activity of saidcancer cells.
 5. A method according to claim 4 wherein the HR dependentDNA DSB repair activity of said cancer cells is determined by measuringthe formation of foci containing Rad51 in the nucleus of the cancercells in response to DNA damaging agents or PARP inhibitors
 6. A methodaccording to claim 1 wherein said cancer cells are identified asdeficient in a HR dependent DNA DSB repair pathway by determining thepresence of one or more mutations or polymorphisms in a nucleic acidsequence encoding a component of HR dependent DNA DSB repair in thecancer cells.
 7. A method according to claim 6 wherein a portion of thenucleic acid sequence encoding a component of HR dependent DNA DSBrepair suspected of containing mutations or polymorphisms is amplifiedusing an amplification reaction and the amplified nucleic acid is thentested to determine the presence or absence of one or more mutations orpolymorphisms which reduces or abrogates the expression or activity ofthe HR dependent DNA DSB repair pathway component.
 8. A method accordingto claim 6 wherein the presence of one or more mutations orpolymorphisms in a nucleic acid sequence encoding a component of HRdependent DNA DSB repair in the cancer cells is determined at theprotein level by detecting the presence of a variant polypeptide whichpolypeptide is a component of HR dependent DNA DSB repair.
 9. A methodaccording to claim 8 wherein the variant polypeptide is detected bycontacting a cancer cell sample with a binding member directed againstthe variant polypeptide.
 10. A method according to claim 9 wherein thebinding member is an antibody molecule.
 11. A method according to claim6 wherein said component of HR dependent DNA DSB repair is selected fromthe group consisting of: ataxia telangiectasia mutated (ATM), RAD51homolog (RecA homolog, E. coli) (S. cerevisiae) (RAD51), RAD51 homolog B(S. cerevisiae) (RAD51L1), RAD51 homolog C (S. cerevisiae) (RAD51C),RAD51 homolog D (S. cerevisiae)(RAD51 L3), DMC1 dosage suppressor ofmck1 homolog, meiosis-specific homologous recombination (yeast) (DMC1),X-ray repair complementing defective repair in Chinese hamster cells 2(XRCC2), X-ray repair complementing defective repair in Chinese hamstercells 3 (XRCC3), RAD52 homolog (S. cerevisiae) (RAD52), RAD54-like (S.cerevisiae)(RAD54L), RAD54 homolog B (S. cerevisiae) (RAD54B), breastcancer 1 (BRCA1), breast cancer 2 (BRCA2), RAD50 homolog (S. cerevisiae)(RAD50), MRE11 meiotic recombination 11 homolog A (S cerevisae)(MRE11A)and nibrin (NBS1).
 12. A method according to claim 11 wherein saidcancer cells are identified as homozygous for a mutation in ATM, RAD51,RAD51 L1, RAD51C, RAD51 L3, DMC1, XRCC2, XRCC3, RAD52, RAD54L, RAD54B,BRCA1, BRCA2, RAD50, MRE11A, or NBS1.
 13. A method according to claim 11wherein said cancer cells are identified as heterozygous for a mutationin ATM, RAD51, RAD51 L1, RAD51C, RAD51 L3, DMC1, XRCC2, XRCC3, RAD52,RAD54L, RAD54B, BRCA1, BRCA2, RAD50, MRE11A, or NBS1.
 14. A methodaccording to claim 1 wherein said cancer cells are breast, ovary,pancreas or prostate cancer cells.
 15. A method according to claim 1wherein said PARP inhibitor is a PARP-1 inhibitor.
 16. A methodaccording to claim 1 wherein said PARP inhibitor is a nicotinamide, abenzamide, an isoquinolinone, a dihydroisoquinolinone, a benzimidazole,an indole, a phthalazinone, a quinazolinone, an isoindolinone, aphenanthridine, a phenanthridinone, a benzopyrone, an unsaturatedhydroximic acid derivative or a pyridazine.
 17. A method according toclaim 16 wherein said PARP inhibitor is a phthalazin-1(2H)-one.
 18. Amethod according to claim 1 wherein the method comprises obtaining saidsample of cancer cells from the individual.
 19. A method of determiningwhether an individual having a cancer condition will respond to PARPinhibitor treatment comprising; contacting a PARP inhibitor with asample of cancer cells obtained from the individual having the cancercondition, and; determining the amount of cell death in said sample,wherein the presence of cell death in said sample indicates that theindividual will respond to said treatment and the absence of cell deathin said sample indicates that the individual will not respond to saidtreatment.